Evaluation of serum concentration of acute‐phase proteins (haptoglobin and serum amyloid A) in the affected Arabian foals with rhodococcosis

Abstract Background Early detection of Rhodococcus equi pneumonia in foals is essential for horse health and for veterinarians. Objectives This study aimed to demonstrate the usefulness of assessing the serum concentration of acute‐phase proteins (APPs) in the early diagnosis of pneumonia. Methods The study evaluated APPs in 19 Arabian foals with R. equi pneumonia and compared them with 18 normal Arabian foals in equestrian clubs in Tabriz, Iran. Affected foals were identified through history, clinical findings and bacterial culture of tracheal washing. Biochemical methods and polymerase chain reaction tests were performed by examining the 16S rRNA and vapA genes to confirm the diagnosis of bacterial isolates. Blood samples were taken from all sick and healthy horses, and their serum was isolated. APPs in the serum were measured in all the samples. Results Rhodococcosis increased the serum concentration of haptoglobin (Hp) and serum amyloid A (SAA) (p < 0.001). The relationship between SAA and Hp was meaningful in the infected group (r = 0.933) but not in the healthy group. In cases where there are clinical findings of R. equi pneumonia, the concentration of SAA and Hp can help the effectiveness of treatment.‐ Conclusions Serum concentration analysis of APPs can be helpful in early diagnosis and successfully treating foals with R. equi pneumonia.

nasal discharge (Reed et al., 2017). Since the disease causes lung abscesses, it is possible not to hear abnormal sounds. It leads to nonpainful polyarthritis in 20% of infected foals and immune-mediated uveitis in 10% of them. It may also form an abdominal cavity and other membrane abscesses. Ultrasound and radiography are effective in the diagnosis of lung abscesses. According to laboratory findings, leukocytosis and increased serum fibrinogen are observed in infected foals, and the serum amyloid A (SAA) level also rises in them (Cohen, 2006). Confirmation of the disease is based on the culture of tracheal secretion and bronchial isolation, which is obtained with the aspiration of the trachea or alveolar lavage. Culturing result is positive in 86% of cases. Polymerase chain reaction (PCR) is another definitive diagnosis that is 100% successful (Constable et al. 2017). This disease must be subtracted from interstitial pneumonia, viral pneumonia, bacterial pneumonia such as Pasturella and Streptococcus pneumoniae and parasitic pneumonia. Rhodococcosis causes many biochemical changes in serum levels, and the study of these changes will help prevent and control the disease; for example, checking the status of acute-phase proteins (APPs) is important. APPs are some plasma proteins that increase or decrease during infection and inflammation. Most of the plasma proteins are synthesised in the liver, although some are synthesised in plasma cells like gama-globulin or endothelial cells. In response to inflammation, these proteins increase (positive APPs) or decrease (negative APPs) plasma concentrations. Haptoglobin (Hp) and SAA are APPs that are important in horses and may increase at the inflammation time. SAA is one of the largest APPs in horses. The concentration of this protein is deficient during a healthy situation but increases rapidly in a few hours (100-1000 mg/L) in the inflammation phase (Browning et al., 2004;Chavan et al., 2005). The main APP in horses is SAA, although Hp is also one of the effective APPs (Cray et al., 2009). Other examples of different proteins in horses that can be measured include fibrinogen, albumin, Hp and α1-glycoprotein. Free haemoglobins pass through the glomerulus and enter the tubules and are usually deposited there. However, the haemoglobin haptoglobin (Hb-Hp) complex is a great complex that cannot pass the glomerulus.
Thus, it seems that haemoglobin is responsible for preventing losses of free Hp from the kidney. This helps maintain haemoglobin's iron (Fe++) (Petersen et al., 2002;Pihl et al., 2013). In horses, studies have been performed on HP in viral diseases and after surgery, and some signs of increased serum HP have been observed in cases of nonseptic arthritis Kent & Goodal, 1991). The increase in plasma concentration SAA is proportional to the amount of damage to the tissue, and the serum SAA level increases within 6 h after inducing acute-phase reaction (De Cozar et al., 2020;Satué et al., 2013). In toxins, SAA isotopes 1 and 2 are usually produced in the liver and secreted into the bloodstream after activating acute-phase reaction. Isotope 3 is found in liver cells, several different tissues, including endothelial lining in the lungs and gastrointestinal tract, and inflamed synovial fluid in horses during acute-phase reaction (Berg et al., 2011;Satué et al., 2013).
This study examined the serum levels of APPs (Hp and SAA) in foals infected with R. equi and compared them with those in healthy foals.

MATERIALS AND METHODS
This study was conducted on 19 infected Arabian foals in Tabriz, Iran, for 3 months. The infection of the foals was confirmed according to clinical signs and laboratory findings.

Tracheal washing
After prescribing a sedative (xylazine 0.5 mg/kg IV), the middle third of the foal's neck was shaved. Next, 2 to 5 ml lidocaine was injected subcutaneously between two tracheal rings aseptically. A small incision (0.5 cm) was then made in the area, and a cannula/needle was inserted at the incision point. Afterwards, 50 ml of 0.9% normal saline was introduced into the trachea with a special catheter, and then aspiration started.

Bacterial culture
Tracheal aspiration samples were transferred to a diagnostic laboratory inside a special tube. In the laboratory, the samples were cultured on a blood agar medium containing 5% sheep blood and incubated at 37 • C for 48 h under aerobic conditions. Mucoid colonies, which are smooth, shiny and non-hemolytic, represented R. equi bacteria, and biochemical and Christie Atkins Munch Peterson (CAMP) tests were used to confirm the diagnosis.

Preparation of DNA from cultured colonies
The isolates were stored in a bacterial laboratory after biochemical approval. For short-term storage for daily experiments, Infusion Agar was cultured linearly and stored in the refrigerator. Glycerin was stored at −70 • C for long-term storage to prevent genetic changes. Pure and isolated biochemical isolates were used for DNA extraction. To this end, the gram-positive bacterial DNA extraction kit of Tissue Genomic DNA Extraction Mini Kit (FavorPrep, Cat No. FATGK001) was used.
Next, a complete loop was transferred from the bacterial colony to a microtube and centrifuged at 13,000 rpm. The bacterial pellet was dissolved in 50 μl of lysozyme reaction solution, vortexed for a few seconds and incubated at 37 • C for 60 min. In the next phase, 20 μl of proteinase K along with 200 μl of FATG2 buffer was added to the sample. Subsequently, the mixture was vertexed, incubated at 60 • C for 30 min and then vortexed occasionally during incubation. Next, 200-μl ethanol 96% was added to the sample mixture. An FATG Mini Column was placed in a collection tube. The mixture was then carefully transferred to the column, centrifuged at 13,000 rpm for 1 min and placed in a new collection tube. Afterwards, 400-μl wash buffer 1 was added to the column, which was then centrifuged at full speed for 1 min and discarded flow-through. Next, 750-μl wash buffer 2 was added to the column, which was then centrifuged at full speed for 1 min and discarded flow-through. The column was centrifuged at full speed to dry for an additional 3 min. Subsequently, 100 μl of preheated elution buffer was added to the membrane of the FATG Mini Column, and the column was centrifuged at full speed for 2 min to elute DNA. The final solution was analysed for DNA quality and quantity using nanodrop and agarose gel. The DNAs were stored at −20 • C for further research.
After extraction, all the DNAs were concentrated using a photometer (BioRad), and their values were checked at optical density (OD) 260/280 and OD 280/230, which were suitable in terms of purity. Also, 5 μl of each DNA were taken on an agarose gel to be tested for quality, which yielded good results.

Molecular testing
All

APP monitoring
A 20-ml blood sample was collected from the jugular vein of each diseased foal and frozen after the serum was separated from each sample.
After separating serum, levels of APPs (Hp and SAA) were measured.
Serum Hp was measured using a specific enzyme linked immunosor- SAA was measured using the ELISA kit (Innovative Research) based on an immunoassay test. SAA reacted with SAA antibodies. After sepa-rating nonbonded proteins with washing, enzyme-conjugated antibodies and the HRP reagent were detected. These enzymes were labelled as antibodies banded with SAA. After washing, the antibody-banded enzyme was measured by adding TMB with a 450-nm wavelength. The level of these enzymes shows the exact SAA level.

Statistical analysis
Twenty samples of R. equi infected foals and 20 healthy foals were selected based on the n =  Figure 2).
The relationship between SAA and Hp was meaningful (r = 0.933) in the infected group but not significant in the control group (Table 2).

DISCUSSION
Rhodococcus equi causes mild to severe respiratory clinical signs in foals. (Reed et al., 2017). Infection from R. equi in foals has been reported in the United States (Muscatello et al., 2007). In a study, plasmid virulence genes of R. equi and its virulence showed that this bacterium could survive a long time in the environment and live tissues (Letek et al., 2008).
Research has found that age, immune suppression and environmental condition have the highest effect as risk factors (Chaffin et al., 2000).
It is an inflammatory process that can cause changes in serum levels of APPs and inflammatory markers.
In foals infected with R. equi, the measurement of SAA was expressed as a diagnostic criterion (Cohen et al., 2005). SAA was measured in an infected horse with influenza and reported as an AAP and inflammatory marker (Hultén, Sandgren, et al., 1999). In another study,  also, the elevation of serum Hp in milk fever has been reported (Skinner et al., 1991). In some studies, serum Hp is used to predict the disease.
Carter et al. noted using Vitamin E and its effect on serum Hp levels in sick animals with a respiratory disorder (Carter et al., 1962). In the present study, the SAA and Hp concentrations significantly increased for foals with R. equi due to inflammatory conditions.
A study was performed on a relatively large population of involved foals and the control group on similar farms. In the study, SAA concentrations were evaluated in 212 horses, and no predictive value was found at the onset of clinical signs of pneumonia (Cohen et al., 2005).
Also, in a study with a smaller population, screening with SAA did not help detect pneumonia caused by R. equi in an involved farm. Only two of the six cases with pneumonia had elevated serum SAA concentrations (Passamonti et al., 2015). Contrary to the above studies, the present study showed that pneumonia caused by R. equi could increase the APPs. SAA is usually rapidly increased due to tissue damage, inflammation or infection, although it can be detected normally in healthy horses (Hultén, Tulamo, et al., 1999). Serum SAA concentrations in foals have been reported to be higher than normal during infection with R. equi or other bacterial infections (Hultén & Demmers, 2002).
Also, the serum SAA concentration in horses was directly related to the clinical signs of respiratory diseases and increased . Along with studies mentioned in the present study, an increase in serum SAA was observed following rhodococcosis. One study found that measuring SAA was as useful as measuring fibrinogen, and it was better than predicting which horse was causing the clinical signs of R.
equi pneumonia. The SAA concentration in this study was significantly higher in pneumonic foals than in healthy foals (Giguère et al., 2016).
Hp usually rises 24 h after inflammation and can be a good diagnostic tool for chronic inflammatory disease (Cray & Belgrave, 2014

CONCLUSION
The findings of this study indicate that R. equi causes increase in the serum level of APPs, Hp and amyloid A, and increasing one serum parameter depends on and drives improvement in the other one.
Therefore, these cases can be considered in treating and controlling the disease. Of course, serial sampling studies are needed to measure these serum parameters. It is also suggested to use additional methods for early detection of pneumonia in foals.

ACKNOWLEDGMENT
The authors would like to thank Tabriz Medical Sciences Branch, Islamic Azad University, for the financial support of this research, which is based on a research project contract.

CONFLICTS OF INTEREST
The authors declare that there is no conflict of interest.

AUTHOR CONTRIBUTIONS
The study was designed by Ali Hassanpour and Sina Moghaddam.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

FUNDING INFORMATION
The authors received no specific funding for this work.

ETHICS STATEMENT
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. Animal experiments were carried out by the guidelines described by the Institutional Animal Care and Use Committee (IACUC).